Papillomavirus infection and male infertility: A systematic review and meta‐analysis

Abstract Background and Aims Increasing attention is being paid to the role of human papillomavirus (HPV) in men and specifically reproduction. Growing evidence suggests an association between HPV infection with many adverse effects including the impairment of semen parameters, the increase of blastocyst apoptosis, the reduction of endometrial implantation of trophoblastic cells, as well as the increase rate of miscarriages and spontaneous preterm birth. Methods We systematically searched PubMed/MEDLINE, Scopus, Embase, Web of Science, CINHAL, PsycINFO, and ERIC from inception to 2nd of July 2024, for studies that investigated the association between HPV infection with sperm parameters and fertility outcomes. The meta‐analysis was conducted on mean data and standard deviations. Results We included 25 studies with a total of 6942 patients. Sperm morphology was lower in HPV positive groups versus HPV negative control groups (SMD = ‐0.52 95% CI −0.84; −0.21; p = 0.001). Sperm motility was also significantly lower in HPV positive groups when compared to HPV negative controls (SMD = −0.82 95% CI −1.07; −0.57; p = <0.001). Sperm volume, concentration, and pH were not significantly different between the two groups. The other 15 studies included in the systematic review for which it was not possible to conduct a meta‐analysis showed strong associations between HPV infection and impairment of sperm parameters, reduced couple fertility and increased risk of pregnancy loss. Conclusions The current evidence highlights the link between HPV infection and sperm parameters, male fertility and reproductive outcomes, which has the potential to lead to a decreased couple fertility, increased risk of pregnancy loss, re‐infection and increased treatment costs.


| INTRODUCTION
Human papillomavirus (HPV) is the most common sexually transmitted viral infection worldwide affecting both males and females. 1 There are more than 200 known subtypes including High-Risk-HPV (HR-HPV) and Low-Risk HPV based on their oncogenic potential. 2It has been shown that most sexually active men and women acquire at least one HPV infection at some point in their lives and, some of them, could be repeatedly infected although most infections remain asymptomatic. 3terestingly, the global prevalence of HPV infection in women with normal cytology is around 11-12%, with the highest prevalence in sub-Saharan Africa at 24%, Eastern Europe 21% and Latin America 16%.
Moreover, HPV prevalence peaks in adolescence and those under 25 years old. 4 Although HPV infection in men is less investigated than in woman, the global prevalence in men is estimated to be ~30% with the infection peak between the ages of 25 and 29 years and a HR-HPV prevalence of 21%. 1 In general, HPV clinical manifestation in men includes anogenital warts and penile, anal, and oropharyngeal cancers and recurrent respiratory papillomatosis. 5,6For women, The World Health Organization (WHO) guidelines recommend using HPV DNA detection every 5-10 years as a primary screening test for those aged between 30 and 50 years and, when HPV DNA is not available, visual inspection after acetic acid application or cytology every 3 years. 7Although sexually active men are at risk of HPV-related morbidity and represent a reservoir for HPV, there is no standardized approach for screening in men. 8However, increasing attention is being paid to the role of HPV in men also due to its role in reproduction.Indeed, growing evidence suggests associations between HPV infection with multiple adverse effects including the impairment of semen parameters, the increase of blastocyst apoptosis, the reduction of endometrial implantation of trophoblastic cells, the increase rate of miscarriages, and spontaneous preterm birth. 9Interestingly, men affected by idiopathic infertility show a higher prevalence of HPV infection, asthenozoospermia and anti-sperm antibodies (ASAs) compared with the general population. 10Moreover, HPV in semen has been demonstrated to also have an impact on reproductive outcomes. 11To date, the role of HPV in male and couple fertility it is not fully understood and the scientific literature addressing male aspects remains less documented than for females.The aim of this review and meta-analysis was to assess associations between HPV infection with sperm parameters and its correlation with couple fertility.

| METHODS
This systematic review adhered to the PRISMA 12 and MOOSE 13 statements and followed a structured protocol registered on PROS-PERO (CRD42024510030).Any inconsistencies were resolved by consensus with a third author (LS).

| Data sources and literature search strategy
In PubMed, the following search strategy was used: ("Human Papillomavirus" OR "Papillomaviridae" OR HPV) AND ("male fertil*" OR "male infertil*" OR "male subfertil*" OR "fertil* men" OR "infertil* men" OR "subfertil* men" OR "Sperm" OR "Sperm quality" OR "Sperm count" OR "Sperm volume" OR "Sperm motility" OR "Sperm vitality" OR "Sperm antibodies" OR "Sperm pH" OR "Sperm viscosity" OR "Sperm morphology" OR "Sperm DNA" OR "sperm DNA fragmentation" OR "Sperm DNA integrity" OR "semen quality" OR "semen parameters").Conference abstracts and reference lists of included articles were hand-searched to identify any potential additional relevant work.

| Study selection
Following the PICOS (participants, intervention, controls, outcomes, study design) criteria, we included studies assessing the influence of HPV infection on sperm parameters and fertility outcomes in observational (case-control, cross-sectional, cohort) studies.
The WHO sperm parameters values were considered as reference values. 14udies were excluded if the data were not analyzable; in vitro studies; if it was not possible to consider separated groups (HPV positive and negative) or if they did not clearly report data regarding sperm parameters or fertility outcomes.No language restriction was a priori applied.

| Data extraction
For each eligible study, two independent investigators (MT and DP) extracted: name of the first author and year of publication, setting, sample size, mean age of the population, mean body mass index (BMI), HPV status and genotype, sperm parameters, and fertility outcomes.Data about matching and method (i.e.propensity score) were planned to be extracted between infected and controls, but no study included this information.Any inconsistencies were resolved by consensus with a third author (LS).

| Outcomes
The primary outcomes considered regarded sperm parameters investigated as sperm count, volume, concentration, motility, vitality, morphology, ASAs, DNA fragmentation, chromatin damage and reproductive outcomes.All parameters were reported in the original papers as mean with standard deviations (SDs).

| Assessment of study quality
Two independent authors (MT and DP) carried out the quality assessment of included studies' using the Newcastle-Ottawa Scale (NOS). 15The NOS assigns a maximum of 9 points based on three quality parameters: selection, comparability, and outcome. 16Any inconsistencies were resolved by consensus with a third author (LS).

| Data synthesis and statistical analysis
All analyses were performed using the meta package inR. 17For all analyses, a two-sided p value less than 0.05 was considered statistically significant.Studies were eligible for inclusion in the meta-analysis if mean data and standard deviations were available for variables in both the H PV+ and HPV-groups.If studies had more than one outcome within the same domain (e.g., two outcomes relating to sperm volume), both the HPV+ and control groups were required to be independent to be included (e.g. the outcomes could not share the same control group).
For all analyses, a random effects meta-analysis using the DerSimonian method 18 was employed, with studies weighted on their inverse variance.Publication bias was assessed using the Egger's test. 19Heterogeneity was assessed using the I 2 statistic.

| Sensitivity analyses
To determine the robustness of results, a sensitivity analysis was performed using the one-study removed method.

| Credibility of evidence
The credibility of all results was classified according to the GRADE criteria, initially based on guidelines proposed by Schünemann et al. 20

| Literature search
As shown in Figure 1, 694 articles were initially screened and 35 full texts were retrieved.Among them, 25  studies were finally included in the systemic review.  The dian quality of the studies was 5.32 (range: 4-7), indicating an overall good quality of the studies, according to the NOS.

| Influence of HPV infection on sperm parameters
Table 1 reports the main information and findings of sperm parameters of included studies.A total of 10 studies with 14 independent outcomes had enough data to be included in the meta-analysis.A total of 10 studies (with 14 independent outcomes) examined sperm motility and sperm morphology, eight studies (nine outcomes) examined sperm concentration, five studies (ten independent outcomes) examined sperm volume, and four studies (eight independent outcomes) examined sperm pH.
Full meta-analysis results can be found in Table 2 and

| Fertility and reproductive outcomes
Table 3 reports the main information and findings on fertility and reproductive outcomes of included studies.Female HPV infection was associated with lower rates of implantation, biochemical pregnancy, and clinical pregnancy as well as with a slightly higher frequency of biochemical miscarriage and clinical miscarriage. 21The presence of HPV infection in sperm impacts negatively on assisted reproduction with a reduction of clinical pregnancies 28,30,45 and increase in pregnancy loss. 23,41

| DISCUSSION
The findings of our systematic review and meta-analysis suggest a role of HPV infection in men both in impairing sperm parameters and in affecting pregnancy outcomes.In terms of sperm parameters, sperm morphology and motility were lower in HPV positive males compared to HPV negative.These results are in line with one previous review focusing on sperm quality. 46  hypothesized for explaining impairment in parameters is likely owing to the presence of HPV itself and the highest presence of ASA. 34deed, previous literature has demonstrated an interaction between HPV with sperm receptors localized in the equatorial region of the sperm head. 47Moreover, the presence of HPV in the sperm is frequently associated with ASAs in infertile patients. 34,38Moreover, ASAs may affect male fertility through sperm agglutination and impaired cervical mucus penetration, complement-mediated sperm injury through the female genital tract, and interference with gametes interaction. 34Considering that autoimmune infertility is often treated by assisted reproduction techniques (ART) and that these procedures can be ineffective in the presence of HPV sperm infection, it is T A B L E 2 Full meta-analysis results.The association between HPV sperm infection and recurrent pregnancy lost The prevalence of HPV sperm infection was significantly higher in couples affected by recurrent pregnancy lost than in their controls.
The semen sample was infected by HPV in approximately 1 out of 5 patients.recommended screening for HPV in all asymptomatic subjects with ASAs.
Studies assessing pregnancy and ART outcomes related to HPV infection are still too limited to perform any statistical analysis, but important considerations can be drawn from existing literature.Indeed, Perino et al. found a significant increased risk of pregnancy loss in couples who underwent ART when HPV was present in sperm. 41Another study reported the reduction in both natural and assisted cumulative pregnancy rate and an increase in miscarriage rate in association with the presence of HPV at sperm level. 35Interestingly, women underwent intra uterine insemination with HPV positive sperm showed 4 times fewer clinical pregnancies compared with those with negative partners. 28cently, another study revealed that the prevalence of HPV sperm infection is significantly higher in couples affected by recurrent pregnancy loss compared to controls. 23Similar results have been observed also in cases of positive women.Indeed, women with HPV infection are associated with lower rates of implantation, biochemical pregnancy, and clinical pregnancy as well as with a slightly higher frequency of biochemical miscarriage and clinical miscarriage. 21,48Again, in one study HPV positive women which underwent intrauterine insemination had six times less pregnancies than those wo were HPV negative. 49veral mechanisms have been hypothesized by which HPV infection can lead to infertility and its negative impact on the success of spontaneous and assisted reproduction could be related to the alteration of sperm parameters, induction of DNA damage, and genomic instability. 50Moreover, in vitro studies found that HPVtransfected trophoblast cells have an increased rate of stage-specific maturation arrest and apoptosis and a reduced placental invasion into the uterine wall compared with control cells. 35Moreover, HPV can compromise trophoblast engraftment and embryo development, leading to pregnancy loss. 9Finally, anti-HPV immune response could cause infertility rejecting the HPV-infected embryo caused by maternal graft-versus-host disease against HPV infected fetus. 9portantly, host immune responses and vaginal microbiome have a natural role in HPV infection, although HPV use different immune evasion mechanisms to limit antiviral activity of immune response inducing tolerance in the host's immune system and reducing the natural clearance. 51dependent from the mechanism, it is urgent to find integrated and multidisciplinary solutions which consider the complexity of reproductive care.Although it is not possible from our results to draw final conclusions, we can make some recommendations.
First, HPV DNA testing, also considering its low cost compared to a failed ART procedure, in male partners of infertile couples, even if asymptomatic, could be useful to find this possible infertility cause and, thus, find the best solution.Considering that the estimated clearance for the virus in sperm is >60% at 6 months, in the case of young couples there is the possibility for waiting for spontaneous healing to restore normal sperm parameters and aim for spontaneous pregnancy or to improve the ART outcomes.Instead, in case of aged couples with no possibility for waiting for spontaneous clearance, specific enzymatic washing procedure of infected semen have been shown to be effective for HPV removal. 33,52Moreover, HPV vaccination has been suggested, showing promising findings, not only in preventing clinical manifestations but also in speeding up the clearance. 53,54In particular, the nonavalent HPV vaccine seems to offer wider protection especially in men with positive HPV partners, which would play a role in the transmission of the infection and relapse.
However, considering that a significant reduction in HPV related infections can be achieved only through vaccination coverage above 80%, extensive vaccination programs should be planned also in a public health economically viable approach. 55 addition to these considerations, counseling should be tailored for infected couples paying particular attention to the hygiene of the reproductive tract and of the hands, strict use of personal underwear and personal towels, a complete abstinence from oral and anal sex, smoking reduction or cessation, the practice of protected intercourse only, and, in the case of HPV-related lesions, the treatment and the monitoring of the genital area. 56ndings from this meta-analysis must be considered in light of its limitations: I) not all studies considered all sperm parameters; II) some important confounding factors, such as male age and environmental exposures were not always noted; and III) despite the clear negative impact on sperm and pregnancy outcomes, the exact pathophysiological mechanisms are not fully understood.
In conclusion, the present work identified associations between HPV and sperm parameters and reproductive outcomes, which has the potential to lead to a decreased couple fertility, increased risk of pregnancy loss, re-infection and increased treatment costs.Counseling and vaccination are two pillars for the management of infected couples, However, it is urgent for reproductive specialists, health professionals, researchers and policy makers to collaborate to develop the best models of HPV management.Such models should be tailored to male and female infected patients to prevent infection transmission, reinfection, speed up clearance, and ultimately to improve and increase reproductive outcomes.

FUNDS
None to declare.
Two investigators (MT and DP) independently conducted a literature search using PubMed/MEDLINE, Scopus, CINAHL, Embase, Psy-cINFO, Web of Science and ERIC from inception until July 2nd, 2024.

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I G U R E 1 PRISMA flow chart.T A B L E 1 Main information and findings of sperm parameters of included studies.

F I G U R E 2
Sperm volume levels in HPV+ populations vs. HPV-controls.F I G U R E 3 Sperm concentration levels in HPV+ populations vs. HPV-controls.F I G U R E 4 Sperm motility levels in HPV+ populations vs. HPV-controls.F I G U R E 5 Sperm pH levels in HPV+ populations vs. HPV-controls.F I G U R E 6 Sperm morphology levels in HPV+ populations vs. HPV-controls.GAROLLA ET AL. | 7 of 11 Main information and findings on fertility and reproductive outcome of included studies.
T A B L E 3